MAMMALIAN PC-12 CELL GENETICALLY-ENGINEERED FOR HUMAN CYTOCHROME-P4502E1 EXPRESSION

The stable expression of the human cytochrome CYP2E1 (P450 alcohol) wa s performed in the mammalian cell line PC-12. This cell line expressed cytochrome b5 (58 +/- 12 pmol/mg microsomal protein vs 528 +/- 80 pmo l/mg in microsomal human liver) and a high level of NADPH: cytochrome P450 reductase (140 +/- 20 nmol - min-1 . mg microsomal protein- 1 vs 68 +/- 48 nmol . min-1 . mg-1 in microsomal human liver). An expressio n plasmid was constructed using the cDNA for the human CYP2E1 mRNA and the Rous sarcoma virus (RSV) promoter. This plasmid was co-transfecte d with the plasmid RSVneo into PC-12 cells. Clones were selected for r esistance to the neomycin analog, G418, and then screened for expressi on of the CYP2E1 isozyme by testing for 6-hydroxylation of chlorzoxazo ne, a specific substrate for CYP2E1. Expression of CYP2E1 was confirme d in one clone, DB-7, by Western blot analysis and by measurement of m onooxygenase activities which were not detectable in PC-12 cells. Chlo rzoxazone 6-hydroxylation, n-butanol oxidation and dimethylnitrosamine N-demethylation were localized in microsomes (62, 60 and 63 pmol . mi n-1 - mg microsomal protein-1, respectively) and were inhibited by car bon monoxide and diethyldithiocarbamate, both inhibitors of P450 enzym es. Although the level of the enzyme activities was about a tenth of t hat measured in human liver microsomes, CYP2E1 expressed in DB-7 cells has catalytic competence similar to human liver CYP2E1. DB-7 cells me tabolized acetaminophen and this metabolic activation was shown to be toxic to these cells by release of lactate dehydrogenase. Construction of recombinant cell lines expressing CYP2E1 provides a useful tool fo r studying the catalytic properties of this enzyme and the consequent cytotoxic effects of substrates metabolized by this enzyme.